In article <4mksmq$9l2 at resunix.ri.sickkids.on.ca>, Randall Willis
<willis at gandalf.psf.sickkids.on.ca> wrote:
> Dima,
>> Unfortunately, what Whatman says is true. I have worked with P11 off
> and on for over 8 years and have found it to be the most intractable
> resin on the market.
well, I used PC for a comparable amount of years for purification of DNA
polymerases from eukaryotic cells and I had very little problems with it.
Let's say that on a PC you should not expect to get high resolution, but
it's extremely useful as a first column for fractionation of crude
extracts if you are looking for a DNA binding protein. I used it in either
20 mM KPO4, pH 7.0, eluting it with a single step at 350 mM KPO4, or in 50
mM TrisHCl, pH 7.5, eluting with a NaCl gradient. Typically, less then 30%
of the total proteins will bind, but more then 70% of the total DNA
polymerase activity is retained. After using, I regenerate it by washing
with 1 M KPO4, pH 7.0 (in the first protocol) or 2M NaCl in 50 mM TrisHCl
pH 8.5 (in the second case). Storage is in 20 mM KPO4, pH 7.0, 0.01%
Sodium Azide. I found reproducible patterns of elution up to 1 year of
storage at 4°C. I strongly recommend, anyway, to check the pH after
prolonged storage, since PC is quite sensitive to pH changes (indeed, you
can elute it also with a pH gradient, even if the resolution is not very
good). If you are using a new batch (i.e. prepared from fresh powder),
after the treatment suggested by the booklet (prolonged washing with
high-/low- pH) I would run a first column with a crude extract with step
elution in high salt, in order to remove completely eventual unspecific
inhibitors. Actually, the most used it is, the best yeld you get. Another
tip...use a column with a diameter of about 1/3 or 1/2 of the lenght. Fat,
short columns are better than long, thin ones with PC, especially if the
volume is large. The opposite is true if you use fast flow, high
resolution cation exchangers like S-Sepharose or SP-Sepharose (that are
also stronger, so keep it in mind if your protein has a highly basic pI).
Hope it helps.